Chimeric vsv-g proteins as nucleic acid transfer vehicles

ABSTRACT

The design and generation of a number of chimeric VSV-G (or VSV-G variants) proteins are used as transfer vehicles to enhance delivery of nucleic acids like plasmid DNA, single and double stranded DNA and RNA, and antisense oligonucleotides into human and animal cells. These chimeric VSV-G protein-nucleic acid transfer vehicles have widespread applications to deliver nucleic acids for exon skipping and gene delivery for gene replacement in human and animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent No.61/984,290, filed Apr. 25, 2014, which is incorporated by referenceherein in its entirety.

FIELD OF THE DISCLOSURE

What is disclosed is a chimeric or fusion protein including a membranetransport domain and a nucleic acid binding domain allowing targeteddelivery of nucleic acids in humans and animals for the treatment ofmedical conditions.

BACKGROUND OF THE DISCLOSURE

The vesicular stomatitis virus G glycoprotein (hereinafter referred toas “VSV-G”) is widely used to pseudotype viral vectors due to its widetropism and stability. These viral vectors facilitate gene transductionin human and animals. The VSV-G proteins, when not associated with anyviral vectors, are also alone capable of forming complexes with nakedplasmid DNA in cell free conditions which can be transfected to cellsthereafter.

The fusogenic G glycoprotein of the vesicular stomatitis virus hasproved to be a useful tool for viral-mediated gene delivery by acting asan envelope protein. Due to its wide tropism, VSV-G has been used as anefficient surrogate envelope protein to produce more stable and hightiter pseudotyped murine leukemia virus (MLV)-based retrovirus andlentivirus-based vectors, all of which have been effectively used forgene therapy. The reason behind this pantropism of VSV remained elusivefor a long period. Recently, it has been found that the VSV enters thecell through a highly ubiquitous low-density lipoprotein (LDL) receptorhaving wide distribution.

However, there are some limitations associated with the use of VSV-G. Itis cytotoxic to producer cells, though the use of tetracycline-regulatedpromoters has helped to overcome this problem. In addition, seruminactivation of VSV-G pseudotyped viruses poses a problem and impedestheir function to some extent in vivo. To overcome the latter problem,VSV-G mutants have been generated which are more thermostable as well asserum-resistant. VSV-G mutants harboring T230N+T368A orK66T+S162T+T230N+T368A mutations exhibited more resistance to seruminactivation and higher thermostability.

Apart from acting as a fusogenic envelope protein for many viralvectors, previous studies showed that purified soluble VSV-G itself canbe inserted into lipid bilayers of liposomes and lipid vesicles in cellfree system in vitro. Additionally, it has been shown that VSV-G canform a complex with naked plasmid DNA in the absence of any transfectionreagent and can thereby enhance the transfection of naked plasmid DNAinto cells. Sucrose gradient sedimentation analysis demonstrated thatVSV-G associates with plasmid DNA and MLV gag-pol particles to formternary complexes that co-sediment with high DNA transfecting activity.This transfection could be abolished by adding antibody for VSV-G.

In eukaryotic cells, heritable genetic material is packaged intostructures known as chromatin consisting of DNA and protein. The basicrepeating unit of chromatin is the nucleosome core, which consists of147 base pairs of DNA wrapped in 1.7 left-handed superhelical turnsaround the surface of an octameric protein core formed by two moleculeseach of histones H2A, H2B, H3, and H4. Histones are highly basicproteins that bind very avidly and non-specifically to nucleic acids.Histones were among the first proteins studied due to their relativeease of isolation and all four histone proteins (H2A, H2B, H3, and H4)can be expressed in bacteria. This has allowed purifying andreconstituting of the histone proteins in cell free systems using welldefined protocols. Though the native histone proteins undergo anextensive array of posttranslational modifications, recombinant histonesdo not undergo posttranslational modifications and can be obtained in ahighly pure form due to their high expression levels.

Single Strand DNA-Binding Proteins (hereinafter referred to as “SSBP”)are ubiquitously expressed and involved in a variety of DNA metabolicprocesses including replication, recombination, damage, and repair.SSBP-1 is a housekeeping gene involved in mitochondrial biogenesis. Itis also a subunit of a single-stranded DNA (ssDNA)-binding complexinvolved in the maintenance of genome stability.

Ribonuclease III (hereinafter referred to as “RNase III”) is an enzymethat is expressed in most of the cells and is involved in the processingof pre-rRNA. It has a catalytic domain and an RNA binding domain that islocated in the C-terminal end of the enzyme. Inhibition of human RNaseIII resulted in cell death suggesting a very important role of thisenzyme.

Gene therapy and exon skipping have served as a means of genetransduction or gene manipulation respectively in humans during the pasttwo decades. Gene therapy and exon skipping were initially developed astherapeutic strategies focused to address detrimental monogeneticdiseases for which there were no available options for treatment, e.g.primary immunodeficiency. These approaches later found widespreadapplication in curing neurodegenerative diseases, cancer, metabolicdisorders, and more.

Gene therapy involves delivery of genes of interest cloned in viralvectors which are capable of producing viruses when transduced in humancells. Despite the continuous improvement of retroviral and lentiviralgene transfer systems for gene delivery during the last many years,there remain severe limitations preventing the development of efficientand safe clinical applications for these systems. These limitationsinclude: their inability to target infection to cells of interest,inefficient transduction, propensity of viral vectors to getincorporated in human genome and create mutations, elicited high immuneresponses, inability to be administered intravenously or subcutaneously,and intramuscular administration that only leads to local delivery ofthe gene. Owing to these limitations, no gene therapy based medicationhas been approved by FDA for use in humans, though there have been manyclinical trials during the past two decades and also many ongoingclinical trials.

Exon skipping is a therapeutic strategy where antisense oligonucleotides(AO) are delivered in humans to modulate splicing of genes resulting inmRNA that either produces functional proteins or blocks theirproduction. AOs are short nucleic acid sequences designed to selectivelybind to specific mRNA or pre-mRNA sequences. Despite the very convincingunderlying principle behind this strategy, only one AO has been approvedby the FDA (Vitravene^(TM), an intraocular injection to inhibitcytomegalovirus retinitis in immunocompromised patients; IsisPharmaceuticals, Carlsbad, Calif.), and this drug is no longer marketed.There are certain limitations associated with the use of AOs includingdifficulty in achieving pharmacologically significant concentrations incells due to biological barriers like endothelial and basement membrane,cell membrane, and sequestration by phagolysosomes.

Further discussion on the subjects of gene transfer and delivery may befound in U.S. Pat. No. 7,531,647 (“Lentiviral Vectors for Site-SpecificGene Insertion”); U.S. Pat. No. 8,158,827 (“Transfection Reagents”); andU.S. Pat. No. 8,652,460 (“Gene Delivery System and Method of Use”) andU.S. patent application Ser. No. 14/635,012 (“Chimeric Dystrophin-VSV-GProtein to Treat Dystrophinopathies”. The disclosures of each of U.S.Pat. Nos. 7,531,647, 8,158,827 and 8,652,460 and U.S. application Ser.No. 14/635,012 are incorporated by reference herein in their entireties.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is a chimeric protein incorporating a transport domainand a nucleic acid binding domain and methods of utilizing thosechimeric proteins for targeted delivery of therapeutic nucleic acids.

In some embodiments, the present disclosure is directed to a chimericprotein comprising VSV-G and a nucleic acid binding protein. In someembodiments, the nucleic acid binding protein is a histone. In someembodiments, the histone is selected from the group consisting of: H2A,H2B, H3, and H4. In some embodiments, the histone is tagged with VSV-Gat the C-terminus. In some embodiments, histone is tagged with VSV-G atthe N-terminus.

In some embodiments, the chimeric protein comprises SEQ. ID NO.: 1, SEQ.ID NO.: 2, SEQ. ID NO.: 3, SEQ. ID NO.: 4, SEQ. ID NO.: 5, SEQ. ID NO.:6, SEQ. ID NO.: 7, or SEQ. ID NO.: 8, and pharmacologically acceptableequivalents thereof. In some embodiments, the chimeric protein comprisesSEQ. ID NO.: 15, SEQ. ID NO.: 16, SEQ. ID NO.: 17, SEQ. ID NO.: 18, SEQ.ID NO.: 19, SEQ. ID NO.: 20, SEQ. ID NO.: 21, or SEQ. ID NO.: 22, andpharmacologically acceptable equivalents thereof. In some embodiments,the chimeric protein includes a sequence having at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity with SEQ. ID NO.: 1, SEQ. ID NO.: 2, SEQ. ID NO.: 3, SEQ. IDNO.: 4, SEQ. ID NO.: 5, SEQ. ID NO.: 6, SEQ. ID NO.: 7, or SEQ. ID NO.:8. In some embodiments, the chimeric protein includes a sequence havingat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% sequence identity with SEQ. ID NO.: 15, SEQ. ID NO.: 16,SEQ. ID NO.: 17, SEQ. ID NO.: 18, SEQ. ID NO.: 19, SEQ. ID NO.: 20, SEQ.ID NO.: 21, or SEQ. ID NO.: 22.

In some embodiments, the nucleic acid binding protein is SSBP-1. In someembodiments, SSBP-1 is tagged with VSV-G at the C-terminus. In someembodiments, SSBP-1 is tagged with VSV-G at the N-terminus. In someembodiments, the chimeric protein comprises SEQ. ID NO.: 9 or SEQ. IDNO.: 10, and pharmacologically acceptable equivalents thereof. In someembodiments, the chimeric protein comprises SEQ. ID NO.: 23 or SEQ. IDNO.: 24, and pharmacologically acceptable equivalents thereof. In someembodiments, the chimeric protein includes a sequence having at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity with SEQ. ID NO.: 9 or SEQ. ID NO.: 10. In someembodiments, the chimeric protein includes a sequence having at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity with SEQ. ID NO.: 23 or SEQ. ID NO.: 24.

In some embodiments, the nucleic acid binding protein is RNase III. Insome embodiments, RNase III is tagged with VSV-G at the C-terminus. Insome embodiments, RNase III is tagged with VSV-G at the N-terminus. Insome embodiments, the chimeric protein comprises SEQ. ID NO.: 11, SEQ.ID NO.: 12, or SEQ. ID NO.: 13, and pharmacologically acceptableequivalents thereof. In some embodiments, wherein the chimeric proteincomprises SEQ. ID NO.: 14, SEQ. ID NO.: 25, or SEQ. ID NO.: 26, andpharmacologically acceptable equivalents thereof. In some embodiments,wherein the chimeric protein includes a sequence having at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with SEQ. ID NO.: 11, SEQ. ID NO.: 12, or SEQ. ID NO.:13. In some embodiments, wherein the chimeric protein includes asequence having at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% sequence identity with SEQ. ID NO.: 14,SEQ. ID NO.: 25, or SEQ. ID NO.: 26.

In some embodiments, the present disclosure is directed to a method oftreating a medical condition in a subject comprising the steps ofproviding a therapeutic compound comprising a chimeric protein includingVSV-G, a nucleic acid binding protein, and at least one nucleic acid,and administering to said subject a pharmaceutically active amount ofsaid therapeutic compound. In some embodiments, the present disclosureis directed to a therapeutic compound comprising a chimeric protein asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter that is regarded as the invention is particularlypointed out and distinctly claimed in the claims at the conclusion ofthe specification. The foregoing and other objects, features, andadvantages of the invention will be apparent from the following detaileddescription taken in conjunction with the accompanying drawings.

FIG. 1 portrays chimeric VSV-G H2A protein fractions purified bySDS-PAGE analysis.

FIG. 2 portrays western blot analysis of the proteins in the purifiedfractions from SDS-PAGE analysis as seen in FIG. 1.

FIG. 3 portrays expression of GFP:HEK 293 cells transfected with eGFPN1plasmid.

FIG. 4A portrays GFP-including plasmid eGFPN1 transfected in HEK293cells using purified VSV-G-H2A protein.

FIG. 4B portrays GFP-including plasmid eGFPN1 transfected in NIH 3T3cells using purified VSV-G-H2A protein.

FIG. 5 portrays a method of treating a medical condition using achimeric protein such as that isolated in FIG. 1.

DETAILED DESCRIPTION

In some embodiments, the present disclosure is directed to a number ofchimeric VSV-G (or VSV-G variants) proteins comprising VSV-G and atleast one nucleic acid binding protein. In some embodiments, theseproteins are used as transfer vehicles to enhance delivery of nucleicacids like plasmid DNA, single and double stranded DNA and RNA, andantisense oligonucleotides into human and animal cells.

VSV-G cloned in expression plasmids, when transfected in cells, formsedimetable vesicles in the absence of any viral components. Thechimeric proteins described here efficiently complex with nucleic acidsin cell free systems and can be used as an effective means fordelivering AOs and genes of interest in human and animal cells. Thisapproach mitigates a number of risks and issues that are associated withgene therapy and exon skipping, i.e. there is no risk of toxicityrelated to viral production or risk of viral genome incorporation andpossible mutations arising as a result. Since the VSV-G proteins enterinto cells via the LDL receptors which are almost ubiquitouslyexpressed, the transduction efficiency of the chimeric VSV-G-nucleicacid transfer vehicle is higher than that achieved by exon-skipping. Thechimeric VSV-G-nucleic acid transfer vehicle consistent with someembodiments of the present disclosure can also replace the currentmechanism of gene therapy. As this proposed chimeric VSV-G-nucleic acidtransfer vehicle does not rely on virus production, it has fewer sideeffects and can be administered subcutaneously. This system can be usedfor gene replacement and can have wide application to cure manydisorders arising from genetic mutations.

In some embodiments, wild-type VSV-G is used in the chimeric protein. Insome embodiments, VSV-G variants are used in the chimeric protein. Insome embodiments, the VSV-G variants include the thermostable and serumresistant mutants of VSV-G, e.g. S162T, T230N, T368A, or combinedmutants T230N+T368A or K66T +S162T+T230N+T368A. In some embodiments,variant VSV-G has at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity with wild-typeVSV-G. As used in the following embodiments, the term “VSV-G” refers toboth wild-type VSV-G and VSV-G variants.

In some embodiments, the chimeric protein of the present disclosure hasat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% sequence identity with the combined sequence of VSV-G+nucleic acid binding protein, with the nucleic acid binding proteintagged with VSV-G at the C-terminus. In some embodiments, chimericprotein has at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity with the combined sequenceof VSV-G+nucleic acid binding protein, with the nucleic acid bindingprotein tagged with VSV-G at the N-terminus. In some embodiments, thechimeric protein comprises a nucleotide sequence that has at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with at least one of SEQ. ID NO.: 1, SEQ. ID NO.: 3,SEQ. ID NO.: 5, SEQ. ID NO.: 7, SEQ. ID NO.: 9, SEQ. ID NO.: 11, SEQ. IDNO.: 13, SEQ. ID NO.: 15, SEQ. ID NO.: 17, SEQ. ID NO.: 19, SEQ. ID NO.:21, SEQ. ID NO.: 23, or SEQ. ID NO.: 25. In some embodiments, thechimeric protein comprises an amino acid sequence that has at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with at least one of SEQ. ID NO.: 2, SEQ. ID NO.: 4,SEQ. ID NO.: 6, SEQ. ID NO.: 8, SEQ. ID NO.: 10, SEQ. ID NO.: 12, SEQ.ID NO.: 14, SEQ. ID NO.: 16, SEQ. ID NO.: 18, SEQ. ID NO.: 20, SEQ. IDNO.: 22, SEQ. ID NO.: 24, or SEQ. ID NO.: 26. In some embodiments, anysuitable mutations, substitutions, additions, and deletions may be madeto the chimeric protein so long as the pharmacological activity of theresulting variant chimeric protein is retained.

In some embodiments, the nucleic acid binding protein is selected fromthe group consisting of H2A histone, H2B histone, H3 histone, H4histone, SSBP-1, RNase III, and combinations thereof.

SEQ. ID NO: 1 is a nucleotide sequence of an H2A histone-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 2 is an amino acid sequence of an H2A histone-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 3 is a nucleotide sequence of an H2B histone-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 4 is an amino acid sequence of an H2B histone-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 5 is a nucleotide sequence of an H3 histone-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 6 is an amino acid sequence of an H3 histone-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 7 is a nucleotide sequence of an H4 histone-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 8 is an amino acid sequence of an H4 histone-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 9 is a nucleotide sequence of an SSBP-1-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 10 is an amino acid sequence of an SSBP-1-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 11 is a nucleotide sequence of an RNase III-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 12 is an amino acid sequence of an RNase III-VSV-G chimericprotein, with VSV-G at the C-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 13 is a nucleotide sequence of a partial RNase III-VSV-Gchimeric protein, with VSV-G at the N-terminus, consistent with someembodiments of the present disclosure.

SEQ. ID NO: 14 is an amino acid sequence of a partial RNase III-VSV-Gchimeric protein, with VSV-G at the N-terminus, consistent with someembodiments of the present disclosure.

SEQ. ID NO: 15 is a nucleotide sequence of an H2A histone-VSV-G chimericprotein, with VSV-G at the N-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 16 is an amino acid sequence of an H2A histone-VSV-Gchimeric protein, with VSV-G at the N-terminus, consistent with someembodiments of the present disclosure.

SEQ. ID NO: 17 is a nucleotide sequence of an H2B histone-VSV-G chimericprotein, with VSV-G at the N-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 18 is an amino acid sequence of an H2B histone-VSV-Gchimeric protein, with VSV-G at the N-terminus, consistent with someembodiments of the present disclosure.

SEQ. ID NO: 19 is a nucleotide sequence of an H3 histone-VSV-G chimericprotein, with VSV-G at the N-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 20 is an amino acid sequence of an H3 histone-VSV-G chimericprotein, with VSV-G at the N-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 21 is a nucleotide sequence of an H4 histone-VSV-G chimericprotein, with VSV-G at the N-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 22 is an amino acid sequence of an H4 histone-VSV-G chimericprotein, with VSV-G at the N-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 23 is a nucleotide sequence of an SSBP-1-VSV-G chimericprotein, with VSV-G at the N-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 24 is an amino acid sequence of an SSBP-1-VSV-G chimericprotein, with VSV-G at the N-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 25 is a nucleotide sequence of an RNase III-VSV-G chimericprotein, with VSV-G at the N-terminus, consistent with some embodimentsof the present disclosure.

SEQ. ID NO: 26 is an amino acid sequence of an RNase III-VSV-G chimericprotein, with VSV-G at the N-terminus, consistent with some embodimentsof the present disclosure.

In some embodiments, the present disclosure is directed to a therapeuticcompound comprising a chimeric protein consistent with those describedin the above-identified embodiments. In some embodiments, as shown inFIG. 5, the present disclosure is directed to a method of treating amedical condition within a subject. In some embodiments, the method oftreating a subject comprises the steps of providing 500 a therapeuticcompound comprising a chimeric protein including VSV-G, a nucleic acidbinding protein, and at least one nucleic acid, and administering 510 tothe subject a pharmaceutically active amount of the therapeuticcompound. In some embodiments, at least one nucleic acid is atherapeutic gene.

EXAMPLE

The following example utilizes a VSV-G-H2A chimeric protein constructedfrom a human histone H2A protein tagged with VSV-G at the N-terminus.The VSV-G-H2A chimeric gene was synthesized using the proprietytechnology from Integrated DNA Technologies, Skokie, IL. The VSV-G-H2Agene was cloned in the mammalian expression vector pTT5 at EcoRI andNotI restriction enzyme sites. The plasmid was prepared and sequencedfor confirmation.

HEK293T cells were passed to ˜70% confluency a day prior to transfection(3× T75 flasks, ˜7.5×10⁶ cells/flask). The following day, the cells inT75 flasks were transfected using Lipofectamine® 2000 (Life TechnologiesCorp., Carlsbad, Calif.) (per T75 flask: 3:1 ratio; 20 ug DNA; and 60 μLLipofectamine® 2000). Flasks were incubated at 37° C. and 5% CO₂overnight. 24 hours after transfection, the conditioned media wasremoved and replaced with fresh media (14 mL/flask). Cells were furtherincubated overnight. Conditioned media was harvested and replaced withfresh media (14 mL/flask) and again incubated overnight. Harvested mediawas then filtered using 0.45 μm filter and stored at −80° C. Thefollowing day, conditioned media was harvested again and filtered using0.45 μm filter. Conditioned media was pooled with media from theprevious day (˜84 mL).

Conditioned media was centrifuged using the Optima® Ultra Centrifuge(with swinging bucket rotor SW32Ti) (Beckman Coulter, Inc., Brea,Calif.) at 25,000 rpm for 2 h at 4° C. (3 centrifuge tubes, ˜28mL/tube). Supernatant was removed and pellets were resuspended in 5 mLPBS per tube. 5 mL of 20% sucrose/PBS cushion plus 5 mL resuspendedpellet was added to a new centrifuge tube. PBS was overlaid to fill thecentrifuge tube. Samples were centrifuged at 25,000 rpm for 6 hours at4° C. Supernatant was removed and each pellet was resuspended in 100 μLPBS (300 μL total volume). An additional 100 μL of PBS was added to eachcentrifuge tube to resuspend any remaining VSV-G-H2A protein (300 μLtotal volume). Protein concentration was measured by A660 Assay.

The chimeric VSV-G H2A protein fractions thus purified were run onpolyacrylamide gels before transfer to nitrocellulose membranes.Proteins were run in 4-15% BioRad TGX™ gel (BioRad Laboratories Inc.,Hercules, Calif.) with BioRad Precision Plus Protein™ markers, at 300 Vfor 21 minutes and then stained with SYPRO®-Orange stain (MolecularProbes, Inc., Eugene, Oreg.), the results of which can be seen atFIG. 1. The contents for each lane found in FIG. 1 are as follows: Lane1: Negative Control—untransfected cells only; Lane 2: molecular weightmarker; Lane 3: M20336-01 (20 μL load); Lane 4: M20336-01 (2 μL load);Lane 5: molecular weight marker; and Lane 6: M20336-02 (20 μL load). TheHEK293 untransfected lane did not stain for any protein while rest ofthe lanes containing the fractions of purified VSV-G-H2A chimericprotein stained for proteins confirming the presence of purifiedproteins in the fractions.

After confirming the presence of the proteins in the purified fractions,proteins were run using the same conditions as described above andtransferred to nitrocellulose membrane. The chimeric VSV-G-H2A proteinwas detected by probing with anti-VSV-G-primary antibody and anti-rabbitHRP secondary antibody. Proteins were transferred to nitrocellulosemembrane using Bio-Rad Trans-Blot® Turbo^(TM). Signal was detected usingthe SNAP id® system (Merck KGAA, Darmstadt, Del.) and SuperSignal® WestPico chemiluminescent substrate (Pierce Biotechnology, Inc., Rockford,Ill.), the results of which can be seen in the western blot shown inFIG. 2. The contents for each lane found in FIG. 2 are as follows: Lane1: molecular weight marker; Lane 2: M20336-01 (20 μL load); Lane 3:molecular weight marker; Lane 4: M20336-01 (2 μL load); Lane 5:molecular weight marker; Lane 6: M20336-02 (20 μL load); Lane 7:molecular weight marker; Lane 8: Negative Control—untransfected cellsonly. A band was detected specific to the size of VSV-G H2A chimericprotein at 75 kD in lanes 2 and 6 containing 204 load of protein. Nobands were detected in lanes 4 and 8 with 2 μL load of purified proteinfraction and non-transfected HEK293 protein fraction. Therefore, thepresence of VSV-G-H2A chimeric protein in the purified fraction wasconfirmed.

In order to evaluate the capacity of the purified VSV-G-H2A chimericprotein to act as nucleic acid transfer vehicle, HEK293 cells and NIH3T3 cells were transfected with green fluorescent protein (GFP)expressing plasmid eGFPN1 utilizing the VSV-G-H2A chimeric protein.Firstly, the eGFPN1 plasmid was transfected in HEK293 cells usingViaFect™ transfection reagent (Promega Corp., Madison, Wis.) to confirmthat GFP was expressed properly. Successful GFP expression is shown inFIG. 3.

To determine whether similar expression of GFP could be seen whenVSV-G-H2A chimeric protein was used as a transfer vehicle, 2 μg ofeGFPN1 plasmid was mixed with 3 μg of VSV-G H2A purified chimericprotein and overlaid in each of HEK293 and NIH 3T3 cells seeded oncoverslips in 6-well plates. Cells were incubated for 48 hours beforeanalysis. To detect whether GFP has expressed, the existing medium inthe cells was aspirated, washed in Dulbecco's phosphate buffered saline(DPBS), and then fixed in 4% paraformaldehyde solution. Cells werewashed again with DPBS a couple of times, stained with4′,6-diamidino-2-phenylindole (DAPI), and then mounted in appropriatemounting medium and viewed under a fluorescence microscope. The resultsof this procedure can be seen in FIGS. 4A and 4B, wherein DAPI stainingdepicts the nucleus and the green fluorescence depicts the GFP.Interestingly, the HEK293 and NIH 3T3 cells in which VSV-G-H2A purifiedchimeric protein was used as a transfer vehicle to transfect eGFPN1plasmid expressed GFP. Therefore, it was concluded that VSV-G-H2Achimeric protein, as well as the other chimeric proteins disclosed inthe present disclosure and functional equivalents thereof, arecandidates for use as nucleic acid transfer vehicles as proposed by thepresent disclosure.

One or more embodiments of the present invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A chimeric protein comprising VSV-G and a nucleicacid binding protein.
 2. The chimeric protein according to claim 1,wherein the nucleic acid binding protein is a histone.
 3. The chimericprotein according to claim 2, wherein the histone is selected from thegroup consisting of: H2A, H2B, H3, and H4.
 4. The chimeric proteinaccording to claim 2, wherein the histone is tagged with VSV-G at theC-terminus.
 5. The chimeric protein according to claim 2, wherein thehistone is tagged with VSV-G at the N-terminus.
 6. The chimeric proteinaccording to claim 4, wherein the chimeric protein comprises SEQ. IDNO.: 1, SEQ. ID NO.: 2, SEQ. ID NO.: 3, SEQ. ID NO.: 4, SEQ. ID NO.: 5,SEQ. ID NO.: 6, SEQ. ID NO.: 7, or SEQ. ID NO.: 8, and pharmacologicallyacceptable equivalents thereof.
 7. The chimeric protein according toclaim 5, wherein the chimeric protein comprises SEQ. ID NO.: 15, SEQ. IDNO.: 16, SEQ. ID NO.: 17, SEQ. ID NO.: 18, SEQ. ID NO.: 19, SEQ. ID NO.:20, SEQ. ID NO.: 21, or SEQ. ID NO.: 22, and pharmacologicallyacceptable equivalents thereof.
 8. The chimeric protein according toclaim 4, wherein the chimeric protein includes a sequence having atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity with SEQ. ID NO.: 1, SEQ. ID NO.: 2, SEQ. IDNO.: 3, SEQ. ID NO.: 4, SEQ. ID NO.: 5, SEQ. ID NO.: 6, SEQ. ID NO.: 7,or SEQ. ID NO.:
 8. 9. The chimeric protein according to claim 5, whereinthe chimeric protein includes a sequence having at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity with SEQ. ID NO.: 15, SEQ. ID NO.: 16, SEQ. ID NO.: 17, SEQ. IDNO.: 18, SEQ. ID NO.: 19, SEQ. ID NO.: 20, SEQ. ID NO.: 21, or SEQ. IDNO.:
 22. 10. The chimeric protein according to claim 1, wherein thenucleic acid binding protein is SSBP-1.
 11. The chimeric proteinaccording to claim 10, wherein SSBP-1 is tagged with VSV-G at theC-terminus.
 12. The chimeric protein according to claim 10, whereinSSBP-1 is tagged with VSV-G at the N-terminus.
 13. The chimeric proteinaccording to claim 11, wherein the chimeric protein comprises SEQ. IDNO.: 9 or SEQ. ID NO.: 10, and pharmacologically acceptable equivalentsthereof.
 14. The chimeric protein according to claim 12, wherein thechimeric protein comprises SEQ. ID NO.: 23 or SEQ. ID NO.: 24, andpharmacologically acceptable equivalents thereof.
 15. The chimericprotein according to claim 11, wherein the chimeric protein includes asequence having at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% sequence identity with SEQ. ID NO.: 9 orSEQ. ID NO.:
 10. 16. The chimeric protein according to claim 12, whereinthe chimeric protein includes a sequence having at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity with SEQ. ID NO.: 23 or SEQ. ID NO.:
 24. 17. The chimericprotein according to claim 1, wherein the nucleic acid binding proteinis RNase III.
 18. The chimeric protein according to claim 17, whereinRNase III is tagged with VSV-G at the C-terminus.
 19. The chimericprotein according to claim 17, wherein RNase III is tagged with VSV-G atthe N-terminus.
 20. The chimeric protein according to claim 18, whereinthe chimeric protein comprises SEQ. ID NO.: 11, SEQ. ID NO.: 12, or SEQ.ID NO.: 13, and pharmacologically acceptable equivalents thereof.